Members of the CREB/ATF family of transcription factors form dimers and bind to cyclic-AMP response elements found in a large number of cellular promoters. The diversity of genes regulated by this large group of transcription factors is reflected in the essential functions of individual factors in fetal survival, neurological development, bone growth, and immune system activation (Rudolph et al. 1998, Proc. Natl. Acad. Sci. USA, 95:4481-4486; Reimold et al. 1996, Nature, 379:262-265; Maekawa et al. 1999, J. Biol. Chem. 274:17813-17819). Recently, an important role in coordinating the timing of hepatocyte proliferation in the regenerating liver was demonstrated for the CREB/ATF family member CREM (Servillo et al. 1998, Genes Dev. 12:3639-3643). In addition, the ATF-3 transcription factor was found to be induced in regenerating liver with the kinetics of an early response gene (Chen et al. 1996, Mol. Cell. Biol. 16:1157-1168).
The functions of a further CREB/ATF family member, XBP-1, have not been defined in detail. This transcription factor is expressed ubiquitously in adults but is mainly found in exocrine glands and bone precursors in the embryonic mouse (Liou et al. 1990, Science 247:1581-1584; Clauss et al. 1993, Dev. Dynamics 197:146-156). In vitro studies have demonstrated downregulation of the XBP-1 gene by BSAP, dimerization of XBP-1 protein with c-Fos, and a decrease in MHC class II gene expression when antisense XBP-1 sequences are introduced into Raji cells (Reimold et al. 1996, J. Exp. Med., 183:393-401; Ono et al. 1991, Proc. Natl. Acad. Sci. USA 88:4309-4312). Recently, the expression of XBP-1 was found to be dramatically increased in hepatocellular carcinomas (Kishimoto et al. 1998, Cell Growth Diff. 9:337-344), although whether this upregulation played a role in the malignant phenotype, or was merely a by-product of it, was not established.
This invention pertain to methods and compositions relating to modulation of hepatocyte growth, plasma cell differentiation and/or T cell subsets by modulation of XBP-1 activity. It has now been discovered that the transcription factor XBP-1 plays a critical role in regulating the growth of hepatocytes, the differentiation of plasma cells and the activity of T cell subsets. The invention is based, at least in part, on the observation that mice lacking XBP-1 have severely impaired hepatocyte development, identifying XBP-1 as a transcription factor essential for hepatocyte growth and, furthermore, that mice lacking XBP-1 are severely impaired in plasma cell generation and exhibit defects in production of Th2 cytokines, identifying XBP-1 as a transcription factor essential for plasma cell differentiation and involved in regulation of T cell subsets. To our knowledge this is the first demonstration of a role for XBP-1 in regulating hepatocyte growth, plasma cell differentiation and T cell subsets.
The invention provides methods for identifying compounds that modulate hepatocyte growth, plasma cell differentiation and/or T cell subsets, methods for modulating hepatocyte growth, plasma cell differentiation and/or T cell subsets using agents that modulate XBP-1 activity (e.g., methods to expand hepatocytes in culture by stimulating XBP-1 activity in the cells such that proliferation of the hepatocytes is stimulated, methods for differentiating plasma cells in culture by stimulating XBP-1 activity in the cells such that differentiation of the plasma cells is stimulated and/or methods for modulating Th2 cytokine production by modulating XBP-1 activity) and methods for diagnosing disorders associated with aberrant hepatocyte growth, plasma cell differentiation and/or T cell subset activity based on assessing a change in the expression of XBP-1 (e.g., the level of expression or the form of XBP-1 expressed).
In one aspect, the invention pertains to methods for identifying compounds that modulate hepatocyte growth, plasma cell differentiation and/or T cell subset activity. In one embodiment, the invention provides a method for identifying a compound that modulates hepatocyte growth, plasma cell differentiation and/or T cell subset activity using an indicator composition comprising XBP-1 protein, wherein a test compound that modulates the activity of XBP-1 is selected and then the effect of this selected compound on hepatocyte growth, plasma cell differentiation or T cell subset activity (e.g., Th2 cytokine production) is assessed. In the method, the indicator composition comprising XBP-1 first is contacted with each member of a library of test compounds. The test compound(s) that modulate the activity of XBP-1 protein are selected and the ability of the selected compound(s) to modulate hepatocyte growth, plasma cell differentiation and/or T cell subset activity (e.g., Th2 cytokine production) is determined. The indicator composition can be, for example, a cell that expresses XBP-1 protein, a cell that has been engineered to express the XBP-1 protein by introducing an expression vector encoding the XBP-1 protein into the cell or a cell free composition. Alternatively, the indicator composition may be a cell or cell-free composition that includes an XBP-1 protein and a target molecule, and the ability of the test compound to modulate the interaction of the XBP-1 protein with a target molecule is monitored. In another embodiment, the indicator composition is an indicator cell, which comprises an XBP-1 protein and a reporter gene responsive to the XBP-1 protein. The level of expression of the reporter gene can be used to determine the ability of a test compound to modulate the activity of XBP-1 protein by producing an indicator cell that contains a recombinant expression vector encoding the XBP-1 protein and a vector comprising an XBP-1-responsive regulatory element operatively linked a reporter gene. The indicator cell is contacted with a test compound and the level of expression of the reporter gene in the indicator cell in the presence of the test compound is determined. By comparing the level of expression of the reporter gene in the indicator cell in the presence of the test compound with the level of expression of the reporter gene in the indicator cell in the absence of the test compound, a compound of interest that modulates the activity of XBP-1 protein can be determined.
In another embodiment of the method for identifying compounds that modulate hepatocyte growth or plasma cell differentiation, a test compound(s) is contacted with a hepatocyte or a plasma cell precursor (B cell) or a T cell deficient in XBP-1 and the effect of the test compound(s) on growth, differentiation and/or activity of the cell is determined, to thereby identify compounds that modulate hepatocyte growth, plasma cell differentiation and/or T cell subset activity via means other than through XBP-1 (i.e., compounds that can xe2x80x9crescuexe2x80x9d the XBP-1-deficient phenotype). For example, a compound that induces growth of XBP-1 deficient hepatocytes or that induces differentiation of XBP-1 deficient B cells into plasma cells or that induces production of a Th2 cytokine(s) by XBP-1 deficient T cells can be selected.
In another aspect, the invention pertains to a method for modulating hepatocyte growth, plasma cell differentiation and/or T cell subset activity by contacting hepatocytes, hepatocyte precursors, plasma cells, plasma cell precursors (B cells), T cells or T cell precursors with a modulator of XBP-1 activity such that the growth of the hepatocytes, differentiation of the plasma cells and/or activity of T cell subsets is modulated. In one aspect, this modulatory method pertains to methods of expanding hepatocytes or plasma cells in vitro, through culture of the cells with a stimulator of XBP-1 activity such that growth of the hepatocytes or differentiation of the plasma cells is stimulated. These in vitro methods allow for the expansion of hepatocytes for use in the treatment of, for example, hepatic injury, hepatic failure due to disease (e.g., viral infections, such as hepatitis) or hepatic failure due to toxins (e.g., cirrhosis of the liver). These in vitro methods also allow for the expansion of plasma cells for use in the treatment of, for example, immunodeficiencies characterized by decreased antibody production, as well for general stimulation of humoral immune responses to pathogens. Furthermore, these in vitro methods allow for modulation of production of Th2 cytokines.
In another aspect, this modulatory method pertains to methods of modulating aberrant hepatocyte growth, plasma cell differentiation and/or T cell subset activity in a subject by administering to the subject a therapeutically effective amount of a specific modulator of XBP-1 activity such that aberrant hepatocyte growth, plasma cell differentiation and/or T cell subset activity in a subject is modulated. In one embodiment, the modulator inhibits XBP-1 activity, for example, an antisense oligonucleotide, an intracellular antibody or a peptide that inhibits the interaction of XBP-1 with another protein, such that hepatocyte growth or plasma cell differentiation in the subject is inhibited, or production of Th2 cytokines is inhibited. Such inhibitory methods may be useful in, for example, hepatocellular carcinoma, multiple myeloma, autoimmune disorders associated with the production of pathogenic autoantibodies (e.g., systemic lupus erythematosus), and disorders associated with excess Th2 cell activity. In another embodiment, the modulator stimulates XBP-1 activity, for example, an expression vector encoding XBP-1, such that hepatocyte growth or plasma cell differentiation is stimulated, or Th2 cytokine production is stimulated, in the subject. Such stimulatory methods may be useful in the treatment of, for example, hepatic injury, hepatic failure due to hepatic disease, hepatic failure due to hepatic toxins, immunodeficiency disorders characterized by decreased antibody production, or disorders associated with deficient Th2 cell activity. Stimulation of plasma cell differentiation also may be useful in, for example, stimulation of humoral responses to pathogens and in increasing the efficiency of vaccinations. In the aforementioned modulatory methods, the modulator can be administered directly to, for example, the liver of a subject, a site of plasma cell differentiation in the subject or a site of T cell subset activity. Alternatively, the modulator can be contacted ex vivo with hepatocytes, hepatocyte precursors, plasma cells, plasma cell precursors, T cells or T cell precursors isolated from a subject, followed by administration of the cells back into the subject.
In another aspect, the invention pertains to a method of diagnosing a subject for a disorder associated with aberrant hepatocyte growth, plasma cell differentiation and/or T cell subset activity by detecting a change in expression of XBP-1 in cells of a subject suspected of having a disorder associated with aberrant hepatocyte growth, plasma cell differentiation and/or T cell subset activity. The expression of XBP-1 in cells of a subject suspected of having the disorder is compared, for example, to the expression of XBP-1 in cells of a control subject without the disorder. The diagnosis for a disorder in a subject is based on a change in expression of XBP-1 (e.g. the level or form of XBP-1) in cells relative to a control subject. Elevated levels of XBP-1 expression, or expression of a more active form of XBP-1 (e.g., a constitutively active mutant form of XBP-1) may be associated with a disorder characterized by increased hepatocyte growth (e.g., hepatocellular carcinoma), plasma cell differentiation (e.g., multiple myeloma or autoimmune diseases characterized by production of pathogenic autoantibodies, such as systemic lupus; erythematosus) or Th2 cell activity (e.g., allergy, cancer, infectious diseases) while reduced levels of XBP-1 expression or expression of an inactive mutant form of XBP-1 may be associated with a disorder characterized by decreased hepatocyte growth, plasma cell differentiation (e.g., immunodeficiency disorders), or Th2 cell activity (e.g., certain autoimmune diseases).
XBP-1 deficient cells and non-human animals (e.g., mice) for use in the screening methods of the invention are also provided. XBP-1 deficient cells, such as hepatocytes, can be obtained from early embryos of XBP-1 deficient animals (prior to lethality in utero). Furthermore, blastocyst complementation can be used to create animals deficient both in XBP-1 and a second gene (e.g., RAG-2), to obtain viable animals in which XBP-1 deficient cells contribute to certain cell compartments, such as the lymphoid system to thereby obtain XBP-1 deficient B cells and T cells. Still further, the invention provides animals having a homozygous disruption in the endogenous XBP-1 gene but that carry an XBP-1 transgene driven by a liver-specific promoter (such as the albumin promoter).
Kits for performing the various methods of the invention are also encompassed by the invention.